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|Threading observed in crystal structures, click here to see the Nature Structural & Molecular Biology paper.
||Bending & Binding in substrate recognition see J. Biol. Chem.||Human FEN1 bound to DNA see Cell paper from Grasby & Tainer labs||E. coli FEN with bound DNA from the Artymiuk Lab see Nucleic Acids Research||Mechanism of FEN inhibitors in Nature Chemical Biology
|Branched DNA has appeared in "The X Files", supposedly implanted into
Agent Dana Scully by aliens so they could track her. Far from being
science fiction, branched DNA plays important roles in biology. We usually think about our DNA as being double-helical in
structure, but in fact, DNA adopts a number of different shapes as
part of normal bilogical processes. Flap
endonucleases, 5' nucleases or 5'-3' exonucleases are some of the
names given to a group of ubiquitous
structure-specific nucleases that can cleave branched DNA, thus restoring the classical double helix.
in all living organisms from bacteria to Homo sapiens (see panel above).
Some viruses even carry genes encoding their own flap endonuclease enzymes. In the
last few years most scientific papers describing these enzymes call them flap
endonucleases (FENs), so that is what we will call them
here. Apart from being essential for all cells (they
participate in DNA replication and repair processes, e.g. see review by Lewis et al 2016), they are also
widely used in biotechnology in genotyping, quantitative PCR,
polymorphism screening and molecular biology.
The DNA Pol1 FEN domain was known as the small fragment and was originally described as having 5'-3' exonuclease activity. However, this is indeed a FEN as can be appreciated from the crystal structures FENs and the Thermus aquaticus DNA PolI.FENs are metalloenzymes, with binding sites for 2 or 3 divalent metal ions (see Syson et al). They bind but do not cut DNA in the absence of a suitable divalent metal ion. These enzymes can use a range of divalent metal cofactors ranging incuding Mg, Mn, Co, Ni, Fe, Ni, Zn and even Cu (see Garforth & Sayers, Feng et al). The core structure consists of a central beta sheet with a number of helices adorning it. The active site contains several conserved carboxylates (mostly aspartic acid residues), a conserved tyrosine and important lysine and arginine residues.
|DNA polymerase I
possesses a flap endonuclease domain in addition to the well known
large) fragment carrying the polymerase and proofreading
The DNA Pol1 FEN domain was known as the small
fragment and was originally described as having 5'-3' exonuclease
activity. However, this is indeed a FEN as can be appreciated from the
crystal structures FENs and the Thermus
aquaticus DNA PolI.FENs are metalloenzymes, with binding
sites for 2 or 3 divalent metal ions (see Syson et al).
They bind but do not cut DNA in the absence of a suitable divalent
metal ion. These enzymes can use a range of divalent metal cofactors
ranging incuding Mg, Mn, Co, Ni, Fe, Ni, Zn and even Cu
(see Garforth & Sayers, Feng et al).
The core structure consists of a central beta sheet with a number of
helices adorning it. The active site contains several conserved
carboxylates (mostly aspartic acid residues), a conserved tyrosine and important lysine
and arginine residues.
There are a number of good reviews on biological roles of the FENs (e.g. Bob Bambara's Ann. Rev. Biochem. or Peter Burgers' JBC review). Basically, at least one FEN is required for cell viability as has been demonstrated in mammalian and bacterial cells. For example FEN knockout mice fail to develop through embryogenesis (Kucherlapati et al) and the FEN domain of PolI is required for cell viability in Streptococcus pneumoniae (Diaz et al). The situation was a little confused regarding bacterial FENs until 2007. For example, Cathy Joyce at Yale showed that the gene encoding DNA PolI (the polA gene) can be deleted in E. coli resulting in bacteria that can grow, albeit slowly on minimal media yet Pol1 was essential for Streptococcus pneumoniae. Joyce also showed that adding back a gene encoding just the FEN-domain of PolI was enough to restore full viability (see Joyce & Grindley, 1984). However, at the time she did not know that many bacteria contain a second FEN-encoding gene (see Allen et al) which I hypothesized might be a backup for the polA-encoded FEN function in 1994 (Sayers, 1994). Indeed, this seems to be the case and late in 2007, Fukushima et al showed that bacteria require at least one functional FEN activity for viability ( Fukushima et al 2007).
So what do FENs do in the cell? They appear to play major roles in processing the remnants of the RNA primers that are used to initiate Okazaki fragment synthesis (so-called lagging strand synthesis), in maintaining genome stability and in DNA repair (e.g. see Greene et al and Lindahl & Wood and the reviews above).
The main activity of the FENs is of course their flap endonuclease activity. In its simplest form FENs can cleave 5' flap or "pseudo Y" structures one nucleotide into the double-stranded region immediately downstream of a single-stranded 5' arm. They can also carry out exonucleolyticactivity on free 5' ends of single-stranded or double-stranded DNA. Single strands of DNA are represented as black lines for simplicity, parallel lines indicate double-helical DNA.
Some FENs can cleave single-stranded closed-circular DNA such as the Taq Polymerase-associated FEN and bacteriophage T5 FEN (also known as T5 D15 5'-3' exonuclease see Sayers & Eckstein). They have also been reported to have "gap specific endonuclease" activity or GEN. The term gap endonuclease (GEN) was coined by Shen and coworkers in 2005 when they observed such an activity in human FEN but such and activity was observed for T5FEN much earlier (Sayers & Eckstein 1991). DNA is represented here as a black line for simplicity, parallel lines represent double-helical DNA.
Images on the left show the structure of the T5FEN molecule and a close-up of the active site. The crystal structure of T5 FEN Ceska, Sayers, Stier and Suck, Nature 1996. PDB code 1EXN. It contains conserved residues that we have mutated in order to ascertain their role in FEN function. For example see our results published in Nature Structural and Molecular Biology, PNAS Dervan, PNAS Garforth.
|Enzyme Product Complex. See Anstey-Gilbert CS et al. It reveals the presence of two very closely spaced Mg ions as well as a potassium ion. Structures with and without DNA were obtained.||See Almalki et al. Shows branched DNA threading through the T5FEN. See
For Papers on FENs by the Sayers' Lab.
for papers on all FENs
|Link to Jon Sayers' University web pages.||Dr Jane Grasby's Pages Kinetics of FENs|
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